Cyclophosphamide (CP) is widely used for treating various kinds of cancer. Because of its high intake, metabolism and excretion, these anticancer medications have been detected in the aquatic environment. There is very limited data on the toxicity and effects of CP on aquatic organisms. The present study aims to assess the toxic effect of CP on certain oxidative stress biomarkers (superoxide dismutase-SOD, catalase-CAT, glutathione peroxidase-GPx, glutathione-GSH, glutathione S-transferases-GST and lipid peroxidation-LPO), protein, glucose, metabolising enzymes (aspartate aminotransferase-AST, alanine aminotransferase-ALT), and ion-regulatory markers (sodium ions-Na⁺, potassium ions-K⁺, and chloride ions-Cl⁻), and histology in the gills and liver of Danio rerio at environmentally relevant concentrations (10, 100 and 1000 ng L⁻¹). Exposure to CP for 42 days led to a significant decrease in SOD, CAT, GST, GPx and GSH levels in the gills and liver tissues of zebrafish. The level of lipid peroxidation in the gills and liver tissues of zebrafish was significantly increased compared to the control group. Chronic exposure significantly changes protein, glucose, AST, ALT, Na⁺, K⁺ and Cl⁻ biomarkers. Fish exposed to different levels of CP showed necrosis, inflammation, degeneration and hemorrhage in the gills and hepatic tissues. The observed changes in the studied tissue biomarkers were proportional to both dose and time. In conclusion, CP at environmentally relevant concentrations causes oxidative stress, energy demand, homeostasis disturbances, and enzyme and histological alterations in the vital tissues of zebrafish. These alterations were similar to the toxic effects reported in mammalian models.